The goal of this project is to understand the distribution and functions of cytoplasmic motors in the axon of neurons. This information is intended to lead to an understanding, at the molecular level, of axonal transport as well as the cytoplasmic organization in the axon. An important current question is how kinesin and dynein are organized on the organelle surface and its microtubule substrate. Quantitative images of kinesin bound to purified, taxol~stabilized microtubules (as described in Project #Z01~NS~02610~10 LN) have provided the first direct evidence for cross~bridging of microtubules by single kinesins, and suggested that kinesin might also translocate microtubules and have a role in microtubule as well as fast axonal transport. These structural methods are now being applied to compare anterograde, kinesin~powered organelles with retrograde, dynein~powered organelles in order to understand how direction of organelle transport is controlled. Endoplasmic reticulum (ER) is another component of axoplasm which interacts with cytoplasmic motors. We have shown by a new dye injection method in cerebellar neurons from brain slices (see report #Z01 NS02841~03 LN) that the ER makes one continuous system throughout the Purkinje neuron. A similar dye method is also being used to investigate movements of the ER is crayfish axons. The bacterial flagellar motor in E. coli has been studied as another example of a motor system than can switch direction of translocation. We recently discovered a new cytoplasmic component of the flagellar motor thought to be involved in directional switching. Biochemical and structural analyses of this cytoplasmic component is expected to lead to an understanding of the directional switching.